Natural and synthetic carboxy polysaccharides as well as their reactive derivatives are utilized in a variety of clinical applications, including the preparation of medical devices. The term “reactive carboxy polysaccharide derivatives” refers to a polysaccharide in which a part or all of the carboxy moieties have been modified into active functional groups, e.g., active esters having higher reactivity with nucleophiles than the corresponding carboxylic acid functionality. The hitherto known active esters of polysaccharides are highly insoluble in water. Their reactivity with hydrophilic nucleophiles including proteins and the like is restricted by the need of aprotic solvents.
U.S. Pat. No. 5,856,299 discloses isolated reactive esters of carboxy polysaccharides prepared in an aprotic solvent. These active esters were suggested for preparing activated polysaccharide-based surfaces which can further bind polypeptides or proteins by a nucleophilic substitution reaction. However, the subsequent conjugation of these isolated active esters with nucleophiles requires the use of an aprotic solvent as well, thus limiting the conjugation reactions to proteins or polypeptides miscible in aprotic solvents. Moreover, in order to obtain an isolated esterified polysaccharide, precipitation is required.
Hyaluronic Acid
Hyaluronic acid (hyaluronate, HA), a glycosaminoglycan, is a ubiquitous component of the extracellular matrix (ECM) of all connective tissues. HA is a linear polysaccharide composed of a disaccharide-repeating unit of N-acetyl-D-glucosamine and D-glucuronic acid linked by β1-4 and β1-3 linkages. HA has a range of naturally occurring molecular weights from several thousands to over 10 million Daltons.
The unique viscoelastic properties of HA combined with its biocompatibility and immunoneutrality has led to its use in a variety of clinical applications such as eye surgery and visco-supplementation of joints. HA is known to specifically bind proteins in the ECM and on the cell surface. These interactions are important for stabilizing the cartilage matrix, in cell motility, in cellular proliferation, in wound healing and inflammation as well as in cancer metastasis. Hyaluronic acid was shown to reversibly bind fibrinogen, and this binding alters the formation kinetics of fibrin gels (LeBoeuf et al., 1986; LeBoeuf et al., 1987).
A variety of chemical modifications and crosslinking strategies of native HA have been explored in order to obtain more mechanically robust and more metabolically stable HA derivatives. The principle targets for chemical modification of HA are the hydroxyl and carboxyl moieties. Modifications via the hydroxyl functional groups are primarily useful for the preparation of crosslinked HA by reactions with bifunctional cross linkers, e.g. divinyl sulfone and diglycidyl ethers (U.S. Pat. Nos. 4,582,865 and 4,713,448).
Modifications of the carboxylic functional groups are useful for the introduction of pendant functionalities, which can further be used to obtain crosslinked products or as sites for covalent attachment of various chemicals, e.g. drugs and biochemical reagents (Li et al., 2004; Shu et al., 2004; Bulpitt and Aeschlimann, 1999). These modifications are made using hydrazides or amines. Activation of HA carboxylic functional groups towards nucleophilic attack by hydrazides or amines in an aqueous media, is mainly performed by the use of water-soluble carbodiimides, particularly 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC). Two major procedures for performing said activation are known in the art. The first, developed by Prestwich et al. is disclosed in U.S. Pat. Nos. 5,616,568 and 5,874,417, Prestwich et al. 1998, and Pouyani and Prestwich, 1994. A second procedure is disclosed in U.S. Pat. No. 6,630,457 wherein HA derivatives with pendant hydrazido, amino as well as other functional groups, are formed. WO 07/102,149 to some of the inventors of the present invention discloses hydrazido derivatives of HA. The disclosures of the aforementioned patents are incorporated by reference in their entirety herein.
WO 00/01733 discloses amide derivatives of hyaluronic acid and methods of preparation thereof. The application further teaches biomaterials prepared from the amide derivatives which can be associated with various polymers, including proteins and polysaccharides. There is neither teaching nor suggestion of a HA-fibrin(ogen) conjugate.
U.S. Pat. No. 5,128,326 discloses drug delivery gels based on cross-linked hyaluronic acid or alternatively, hyaluronic acid and a hydrophilic polymer either polysaccharide, protein or glycoprotein. The drug may be dispersed within the gel or may be covalently attached to either of the HA or hydrophilic polymer. A recently published international application WO 07/026,362 discloses a method of preparing cross-linked polysaccharide matrices by cross-linking amino functionalized polysaccharides including amino functionalized HA with reducing sugars and/or sugar derivatives. The resulting matrices include polysaccharides cross-linked with proteins and/or polypeptides. There is neither teaching nor suggestion of a soluble HA-fibrin(ogen) conjugate.
U.S. Pat. Nos. 5,760,200, 6,030,958, 6,174,999 and 6,943,154 disclose water insoluble HA-based biocompatible compositions, formed in an aqueous medium. These compositions were prepared by combining: (a) a polyanionic polysaccharide (b) at least 1 molar equivalent of a nucleophile per molar equivalent of the polyanionic polysaccharide, and (c) at least 0.1 molar equivalent of an activating agent per molar equivalent of said polyanionic polysaccharide, in a “one pot reaction”.
Hyaluronic acid is easily and readily crosslinked, thereby allowing the formation of heterogeneous hyaluronic acid compounds. U.S. Pat. No. 5,972,385 discloses a lyophilized crosslinked collagen-polysaccharide matrix for tissue repair in which collagen is covalently bound to periodate-treated polysaccharide having free aldehyde groups. The crosslinked collagen-polysaccharide forms a slurry, which is poured into a mold and lyophilized to form a sponge. A collagen-polysaccharide matrix further comprising fibrin is disclosed as well.
U.S. Pat. Nos. 6,503,527 and 6,699,484 disclose a fibrin sealant or fibrin adhesive composition comprising fibrinogen, a fibrinogen-cleaving agent and a biomaterial which is a hyaluronic acid material, a chitin material or a chitosan material wherein both the fibrinogen and the fibrinogen-cleaving agent are incorporated on the biomaterial. According to these disclosures, the HA or HA derivatives can be produced according to methods known in the art for derivatizing HA; active esters of HA are neither taught nor suggested. Moreover, no methods whatsoever are disclosed for forming any chemical conjugates between HA and fibrinogen. Thus, the above disclosures neither teach nor suggest a water-soluble polysaccharide-fibrinogen conjugate having a plurality of amide bonds between the carboxylic functional groups of the polysaccharide and the amino functional groups of the fibrinogen.
A water-soluble conjugate of sodium hyaluronate with superoxide dismutase (SOD) was reported by Sakurai et al. (1997). This conjugate showed improved anti-inflammatory activity in vivo. Its water solubility might be attributed to the low molecular weight of bovine SOD, which infers on the physicochemical properties of the conjugate.
Fibrin
Fibrinogen is a major plasma protein, which participates in the blood coagulation process. Upon blood vessel injury, fibrinogen is converted into insoluble fibrin, which serves as the scaffold for a clot. Blood coagulation is a complex process comprising the sequential interaction of a number of plasma proteins, in particular of fibrinogen (factor I), prothrombin (factor II), factor V and factors VII-XIII. Other plasma proteins such as Von Willebrand factor, immunoglobulins, coagulation factors and complement components also participate in the formation of blood clots.
Many fibrin(ogen) containing sealants, clots or scaffolds are known in the art. Fibrin is often used as a tissue adhesive medical device for wound healing and tissue repair. Lyophilized plasma-derived protein concentrate (comprising fibrinogen, Factor XIII and fibronectin), in the presence of thrombin and calcium ions forms an injectable biological sealant (fibrin glue). U.S. Pat. No. 5,411,885 discloses a method for embedding and culturing tissue employing fibrin glue.
U.S. Pat. No. 4,642,120 discloses the use of fibrinogen-containing glue in combination with autologous mesenchymal or chondrocytic cells to promote repair of cartilage and bone defects. U.S. Pat. No. 5,260,420 discloses a method for preparation and use of biological glue comprising plasma proteins for therapeutic use. U.S. Pat. No. 6,440,427 teaches an adhesive composition mainly composed of fibrin forming components and a viscosity enhancing polysaccharide such as hyaluronic acid.
U.S. Pat. No. 5,631,011 teaches a tissue treatment composition to promote wound healing and reduce scar formation consisting essentially of (a) a fibrin glue component comprising fibrin or fibrinogen, Factor XIII, thrombin, bivalent calcium, and (b) a hyaluronic acid component selected from hyaluronic acid, crosslinked hyaluronic acid, or a salt thereof. According to this disclosure, the hyaluronic acid component is present in an amount sufficient to form a viscous composition.
U.S. Pat. No. 5,763,410 discloses the use of kits for the preparation of a fibrin sealant containing fibrin monomer which can be polymerized to form a fibrin sealant when combined with a second component which is distilled water or an alkaline buffer.
U.S. Pat. No. 6,074,663 discloses a cross-linked fibrin sheet-like material for the prevention of adhesion formation. PCT application WO 00/51538 discloses a bioadhesive, porous PEG-crosslinked albumin and fibrin scaffold, useful for wound healing. A freeze-dried fibrin antibiotic clot for the slow release of an antibiotic is described by Itokazu et al. (1997).
A freeze-dried fibrin web for wound healing has been disclosed in U.S. Pat. Nos. 6,310,267 and 6,486,377. A fibrin sponge containing a blood clotting activator for hemostasis, tissue adhesion, wound healing and cell culture support is disclosed in WO 99/15209. WO 04/067704 of one of the applicants of the present invention discloses a porous freeze-dried fibrin matrix which incorporates glycosaminoglycans and bioactive agents for use as an implant for tissue engineering.
There is neither teaching nor suggestion of a polysaccharide-fibrinogen covalent conjugate in any of the above references.
Tissue Engineering
Tissue engineering is defined as the art of reconstructing or regenerating mammalian tissues, both structurally and functionally. It generally includes the delivery of a synthetic or natural scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to repair a wound or defect.
An example of a tissue that is prone to damage by disease and trauma is the articular cartilage, one of several types of cartilage in the body, found at the articular surfaces of bones. Damaged cartilage is amenable to repair.
Matrices useful for tissue regeneration and/or as biocompatible surfaces for tissue culture are well known in the art. These matrices may be considered as substrates for cell growth either in vitro or in vivo. Suitable matrices for tissue growth and/or regeneration include both biocompatible and biostable entities. Among the many candidates that may serve as useful matrices claimed to support tissue growth or regeneration are gels, foams, sheets, and porous structures of different forms and shapes.
Many natural polymers have been disclosed as useful for tissue engineering or culture, including various glycoproteins and glycosaminoglycans (GAGs) of the extracellular matrix, for instance fibronectin, various types of collagen and laminin, keratin, fibrin and fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate and others. U.S. Pat. Nos. 6,425,918 and 6,334,968 disclose a freeze-dried bioresorbable polysaccharide sponge and its use thereof as a matrix or scaffold for implantation into a patient.
There remains an unmet need for water-soluble carboxy polysaccharide derivatives useful per se and in the preparation of soluble polysaccharide-fibrinogen conjugates having utility in tissue engineering, repair and regeneration.